Tonic and acute nitric oxide signaling through solubleguanylate cyclase is mediated by nonheme nitricoxide, ATP, and GTPStephen P. L. Cary†‡, Jonathan A. Winger‡§, and Michael A. Marletta†§¶�††
Departments of †Biological Chemistry and §Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48109; ¶Departments of Chemistry andMolecular and Cell Biology, University of California, Berkeley, CA 94720; and �Division of Physical Biosciences, Lawrence Berkeley NationalLaboratory, Berkeley, CA 94720
Communicated by Jack E. Dixon, University of California at San Diego, La Jolla, CA, July 22, 2005 (received for review May 27, 2005)
Nitric oxide (NO) affects many physiological systems by activatingcGMP signaling cascades through soluble guanylate cyclase (sGC).In the accepted model, NO binds to the sGC heme, activating theenzyme. Here, we report that in the presence of physiologicalconcentrations of ATP and GTP, NO dissociation from the sGC hemeis �160 times slower than the rate of enzyme deactivation in vitro.Deactivated sGC still has NO bound to the heme, and full activationrequires additional NO. We propose an activation model where, inthe presence of both ATP and GTP, tonic NO forms a stable hemecomplex with low sGC activity; acute production of NO transientlyand fully activates this NO-bound sGC.
heme � nucleotide regulation
N itric oxide (NO) mediates blood vessel relaxation, complexaspects of myocardial function, perfusion and function of all
major organs, synaptic plasticity in the brain, platelet aggregation,skin function, and numerous other physiological processes, bytargeting and activating soluble guanylate cyclase (sGC) (reviewedin refs. 1–5). Dysregulation of NO signaling, then, contributes tomany types of disease state, from erectile dysfunction and heartdisease, to neurodegeneration, stroke, hypertension, and gastroin-testinal disease, to name a few (reviewed in refs. 6–8).
In vivo and ex vivo tissue studies of NO have revealed twofundamentally distinct and paradoxical signaling modes: tonic andacute. Tonic NO describes the continual low-level production ofNO that elicits a long-lasting low-level cGMP signal (9). Underresting conditions such as normotension, inhibition of nitric oxidesynthase (NOS) or sGC results in vasoconstriction; therefore, tonicNO-elicited cGMP production maintains homeostatic vasculartone. Relaxation of smooth muscle requires an acute burst of NOsynthesis, typically triggered by acetylcholine, and cGMP levels riserapidly (10). These data, which describe two separate effects of NO,cannot be explained by the established binary model for NOregulation of sGC: that NO activates sGC solely by binding to itsheme cofactor.
The N-terminal �180 aa of the �1 subunit of sGC form anevolutionarily conserved protoporphyrin-IX heme domain withspectral properties similar to the holoenzyme (11–13). NO bindingto the sGC heme is diffusion-limited; however, oxygen does notbind to sGC, and carbon monoxide (CO) binding is at least 106-foldweaker than NO. Thus, the sGC heme environment is a specific NOsensor. The C-terminal domains of each subunit are homologous tothe catalytic domains of adenylate cyclase and fold together to formthe active site of the enzyme (14). In the existing activation model(Fig. 1, black scheme), NO binds to the heme of sGC (15), forminga six-coordinate intermediate (16–18). The rate of conversion ofthis intermediate to the final five-coordinate ferrous-nitrosyl spe-cies depends on the concentration of NO; thus, nonheme NOaccelerates rupture of the proximal histidine–iron bond. Breakingof this bond is thought to result in a conformational change in thecatalytic domain of the enzyme, accelerating the basal rate ofconversion of GTP to cGMP several hundred fold (17, 19, 20).
Although the phenomena of tonic and acute effects of NO�cGMP are widely acknowledged, the in vivo data are clearlyinconsistent with the in vitro binary model for sGC activation byNO. In vivo data suggest that NO is both a long-lasting partialagonist and a transient full agonist of sGC, whereas the in vitrobinary model suggests that NO simply switches the enzyme betweeninactive and fully active states. The sGC heme-NO complex isextremely stable, inconsistent with a rapid deactivation profilein vivo. Recent data also suggest that full activation of sGC by NOat the heme requires high levels of the cyclase reaction products[cGMP and�or pyrophosphate (PPi)] before NO binding (21).Furthermore, ATP at physiological concentrations inhibits sGCactivity by as much as 50% (22, 23). Clearly, NO and nucleotidesaffect sGC function in ways not previously appreciated. We havestudied the activation and deactivation of sGC in the presence ofphysiological concentrations of ATP (�1 mM) and GTP (�200�M) (24), and have uncovered two distinct modes of NO activation.One activation state is low and stable and is due to NO binding tothe heme. An acute transient activation state of the enzymedepends on nonheme NO. Together, these two states support amodel that explains, under physiologically relevant nucleotidelevels, how NO activates sGC to effect the tonic and acute NOsignaling modes observed in vivo.
Materials and MethodsExpression and Purification of Recombinant His-Tagged sGC. Briefly,a C-terminal His-tag was added to the �1 subunit of rat lung sGC.The Bac-to-Bac baculovirus expression system (Invitrogen) wasused to generate recombinant � and � baculoviruses according tothe manufacturer’s protocol. Sf9 cells (American Type CultureCollection) were cultured in Ex-Cell 420 insect serum-free medium(JRH Biosciences) supplemented with 10% FCS (HyClone) and1% antibiotic-antimycotic (Invitrogen). For protein expression,1-liter cultures were coinfected with H6�1 and �1 baculoviruses andharvested after 3 days in an orbital shaker at 28°C. sGC was purifiedto homogeneity by using Ni affinity chromatography and anionexchange chromatography. Purified sGC (exhibiting an A278�A431 �1.1) was concentrated to 5–10 �M in a Vivaspin 6 50k filter (VWRScientific), flash-frozen, and stored in liquid N2. Protein purity wasassessed by SDS�PAGE and was routinely �95%. Protein concen-trations were calculated from the A431 by using an extinctioncoefficient of 148,000 M�1�cm�1 (25).
Abbreviations: �2(1–217), residues 1–217 of the soluble guanylate cyclase �2 isoform;DEA�NO, diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate; EIA, en-zyme immunoassay; GMPCPP, guanosine 5�-[�,�-methylene]triphosphate; NO, nitric oxide;PROLI�NO, disodium 1-(hydroxy-NNO-azoxy)-L-proline; sGC, soluble guanylate cyclase.
‡S.P.L.C. and J.A.W. contributed equally to this work.
††To whom correspondence should be addressed at: Department of Chemistry, Universityof California, 211 Lewis Hall, Berkeley, CA 94720-1460. E-mail: [email protected].
Preparation of Heme Protein NO Traps. Horse heart myoglobin (Mb)was dissolved in 50 mM Hepes (pH 7.4) and 50 mM NaCl, reducedwith sodium dithionite (Na2S2O4) in an anaerobic chamber (CoyLaboratory Products), desalted on a PD-10 column (AmershamPharmacia), and exposed to air to form oxymyoglobin (MbO2). The�2(1–217) (residues 1–217 of the sGC �2 isoform) H-NOX domainconstruct was expressed in Escherichia coli and purified by anion-exchange and gel-filtration chromatography. Plasmid encoding theMycobacterium bovis truncated hemoglobin (HbN) was a gift fromMichel Guertin (Laval University, Quebec, Canada). HbN wasexpressed and purified as described in ref. 26.
Dissociation of NO from the sGC Heme. The dissociation of NO fromthe sGC heme at 10°C was measured by using the CO�Na2S2O4trapping method described in ref. 27. A solution of Na2S2O4 in 50mM Hepes (pH 7.4) and 50 mM NaCl was prepared in an anaerobicchamber and saturated with CO (Matheson) by bubbling for 10 min.Disodium 1-(hydroxy-NNO-azoxy)-L-proline (PROLI�NO) stockswere prepared in 10 mM NaOH and kept on ice. sGC-NO wasprepared by the addition of a small excess of PROLI�NO in theabsence or presence of nucleotide as indicated, in 50 mM Hepes(pH 7.4), 50 mM NaCl, 20 mM MgCl2, and 2 mM DTT at 25°C.sGC-NO (100 �l at 1.6 �M) was placed in an anaerobic cuvette, andthe headspace was replaced with argon. The cuvette and trapsolutions were equilibrated at 10°C for 1 min, and the reaction wasinitiated by the addition of 300 �l of CO�Na2S2O4 solution, usinga Hamilton gas-tight syringe. The final reaction concentrationswere 30 mM Na2S2O4, 400 nM sGC-NO, 5 mM MgCl2, 1 mM ATP,and 200 �M GTP or guanosine 5�-[�,�-methylene]triphosphate(GMPCPP). Any remaining excess NO was immediately destroyedby the vast excess of Na2S2O4. The time from addition of trap toinitiation of data collection was �15 s. Electronic absorptionspectra were collected with a Cary 3E spectrophotometer at 15nm�s. A buffer baseline was subtracted for each spectrum, andspectra were corrected for baseline drift by normalization to theaverage absorbance from 448 to 450 nm. Difference spectra wereobtained by subtraction of the time 0 spectrum for the samplecontaining no nucleotide from all subsequent spectra. �A423 valueswere extracted from the difference spectra and plotted versus timefor each experiment.
Deactivation of sGC-NO by the NO Trap �2(1–217). Deactivationreactions were carried out at 22°C as follows. A small excess ofPROLI�NO was added to sGC in 50 mM Hepes (pH 7.4), 50 mMNaCl, 5 mM MgCl2, and 2 mM DTT (with or without 715 �M ATPand�or 37 �M GMPCPP) in 140 �l. Deactivation was initiated bythe addition of 20 �l of �2(1–217) in 50 mM Hepes (pH 7.4), 50 mMNaCl, 2 mM DTT, and 15 mM GTP to the reaction mixture. Thefinal concentrations were 48.3 �M �2(1–217), 230 nM sGC-NO, 4.4mM MgCl2, 1.9 mM GTP, and, where indicated, 625 �M ATP and32 �M GMPCPP. Aliquots (20 �l) were withdrawn at the indicated
time points and quenched by addition to 480 �l of 125 mMZn(CH3CO2)2, to which was then added 500 �l of 125 mM Na2CO3.The cGMP formed was quantified with a cGMP enzyme immu-noassay (EIA) kit (EIA Format B, Biomol), per the manufacturer’sinstructions. The decline in enzyme activity (as a percentage ofinitial activity) was obtained by fitting the accumulation of cGMPto Eq. 1 as described in ref. 28:
cGMP(t) � �t1�2
ln2(activitymax � activitybasal)(e
�ln�2�tt1�2 � 1)
� activitybasalt . [1]
Data were replotted according to Eq. 2 describing a decline inenzyme activity:
Effect of Nucleotides on the Activity of Ferrous-Nitrosyl sGC. To makeferrous-nitrosyl sGC, aliquots of diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA�NO) were added tosGC in the absence or presence of 30 �M GMPCPP and�or 1 mMATP, and the reaction was monitored by electronic absorbancespectroscopy. Addition of DEA�NO was continued until only asmall shoulder at 431 nm remained, at which point the protein wasassayed. In some experiments, PROLI�NO [t1/2 1.8 s at 37°C (29)]was used; no difference was observed when using this compound.Full conversion to 399 nm was not attained to ensure that no excessNO existed in solution. Using the extinction coefficients of sGC andferrous-nitrosyl sGC, we generated simulated spectra of mixedpopulations and overlaid them on observed spectra for eachaddition of NO to sGC (Fig. 6, which is published as supportinginformation on the PNAS web site). This allowed us to calculate thepercentage of ferrous-nitrosyl sGC in each mixture. Assays in thespectrophotometer were initiated by addition of GTP, mixing, andinitiation of data collection. Spectra were acquired every minutethroughout all assays, which contained 400 nM sGC, 6 mM MgCl2,and 3 mM GTP. After 5–6 min, the reactions were quenched by thewithdrawal of 100 �l from the cuvette and addition to 400 �l of 125mM Zn(CH3CO2)2, to which was then added 500 �l of 125 mMNa2CO3. The cGMP formed was quantified by EIA.
Activation of Ferrous-Nitrosyl sGC by Excess NO. Assays were con-ducted in the spectrophotometer at 22°C as follows. sGC (400 nM)in 50 mM Hepes, 2 mM DTT, and 5 mM MgCl2 (pH 7.4) waspremixed in a cuvette with or without 30 �M GMPCPP andincreasing concentrations of ATP (0 �M, 375 �M, 750 �M, 1.5 mM,or 3.3 mM). Stoichiometric PROLI�NO was added to the sGC�nucleotide mix for 1 min, and then 2-min assays were initiated bythe addition of GTP to 1.5 mM. Spectra were acquired every 30 sthroughout the course of each assay. For each assay, the finalconcentration of ferrous-nitrosyl sGC was determined by compar-ison to calculated spectral mixtures of sGC and ferrous-nitrosyl sGCas above. Experiments were conducted in triplicate, and cGMPaccumulation was measured by EIA. For experiments with excessNO, assays were performed as above, except that the sGC concen-tration was 67 nM, and 1.5-ml Eppendorf tubes were used.
Effect of Nucleotides on NO Binding to the sGC Heme. sGC (400 nM)with or without ATP and�or GMPCPP in 50 mM Hepes (pH 7.4),2 mM DTT, and 5 mM MgCl2 was placed in a cuvette at 22°C. Thesame substoichiometric amount of PROLI�NO was added to eachsGC�nucleotide mix. Electronic absorption spectra were acquiredafter 1 min, corrected for baseline drift, and overlaid for compar-ison of ferrous-nitrosyl sGC formation, as determined by absor-bance increase at 399 nm and decrease at 431 nm. To examine the
Fig. 1. Models for NO activation of sGC. In the scheme depicted in black, NObinds rapidly to the basally active five-coordinate ferrous heme, forming asix-coordinate ferrous-nitrosyl intermediate. At a rate that depends on NOconcentration, the final five-coordinate complex is activated several hundred-fold. In the scheme depicted in red, the modulation of the formation anddissociation of the sGC heme-NO complex is shown, as well as the activationstate of ferrous-nitrosyl sGC, by ATP, GTP, and NO.
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rates of Fe–His bond cleavage in the absence and presence ofnucleotides, sGC (400 nM) in 50 mM Hepes (pH 7.4), 2 mM DTT,and 5 mM MgCl2 was premixed at 4°C with 0 �M or 200 �M GTPand 0 �M, 500 �M, or 3 mM ATP. PROLI�NO (2 �M) was added,and spectra were collected every 12 s.
Effect of YC-1 on the Activation of sGC by NO. Ferrous-nitrosyl sGCwas prepared by the addition of DEA�NO to obtain mostlyferrous-nitrosyl sGC, as described above. YC-1 stocks were madeup in DMSO. In all assays, the final concentration of DMSO was1%, which does not significantly affect enzyme activity. Assays werecarried out in duplicate at 22°C as follows. A 50-�l aliquot ofenzyme (sGC, ferrous-nitrosyl sGC, or ferrous-nitrosyl sGC plusexcess NO) with or without YC-1 was added to 50 �l of 50 mMHepes (pH 7.4), 50 mM NaCl, 5 mM MgCl2, and 1.6 mM GTP.Final concentrations were 200 nM sGC, 1.5 mM MgCl2, 800 �MGTP, and 100 �M YC-1, in a reaction volume of 100 �l. Assayswere quenched, and cGMP was quantified by EIA. Final ferrous-nitrosyl sGC concentrations were determined by comparison tocalculated spectral mixtures of sGC and ferrous-nitrosyl sGC asbefore. The effect of YC-1 on the deactivation of sGC activated byexcess NO was also investigated. sGC with or without YC-1 in 50mM Hepes (pH 7.4) and 50 mM NaCl was activated with DEA�NO(4 �M) in 90 �l. An aliquot (10 �l) of an NO trap solution,containing HbN as the NO trap, MgCl2, and GTP, was added to thesGC-NO solution. The final assay concentrations were 67 nM sGC,5 �M HbN, 5 mM MgCl2, 3 mM GTP, and 100 �M YC-1. Assayswere quenched, and cGMP was quantified by EIA.
Miscellaneous. The NO donors DEA�NO and PROLI�NO werefrom Cayman Chemical. No difference was observed for anyexperiment when NO gas was used instead of a NO donor. Thenoncyclizable GTP analogue GMPCPP was from Jena Bioscience.All other chemicals were from Sigma unless otherwise stated.
Results and DiscussionWe first analyzed rates of NO dissociation from the sGC heme.sGC-NO was produced by addition of a small excess of the NOdonor PROLI�NO to sGC in the presence of GTP, ATP, or bothnucleotides; Mg2 was always present. To remove excess NO,prevent rebinding, and chemically destroy dissociated NO, a CO�dithionite trap was used (27, 30). In the absence of nucleotide, orin the presence of ATP, the NO-heme off-rate is extremely slow(0.00031 � 0.00002 s�1; t1/2 37 min). In the presence of thesubstrate GTP, the NO-heme off-rate is rapid (0.18 � 0.01 s�1;t1/2 3.9 s); however, when both ATP and GTP are present, theNO-heme off-rate is again extremely slow (0.00031 � 0.00002 s�1;t1/2 37 min) (Fig. 2A). The same rates are observed when anoncyclizable analog of GTP (GMPCPP) is used (data not shown).Thus, GTP, in a turnover-independent fashion, accelerates the NOoff-rate from the sGC heme by �500-fold, and ATP blocks thisGTP effect. Intriguingly, the GTP effect occurs only if it is presentwith sGC before NO addition, or if it is added to sGC in thepresence of excess NO. GTP has no effect on the NO-heme off-rateif it is added simultaneously with the NO trap (data not shown). Therapid dissociation of NO in the presence of GTP would beconsistent with the rapid deactivation rates observed in vivo.However, physiological concentrations of ATP block the ability of
Fig. 2. The rate of dissociation of NO from the sGC heme does not correlate with the rate of enzyme deactivation. (A) NO-heme dissociation rate at 10°C inthe absence or presence of nucleotides. sGC was premixed with 200 �M GTP and�or 1 mM ATP, and excess NO was added via PROLI�NO. A CO�dithionite trapwas used to destroy excess and dissociated NO. The increase in absorbance at 423 nm, because of formation of the heme-CO complex, is plotted versus time. Datafrom duplicate runs were averaged and fit to a single exponential to obtain rate constants. (B) Deactivation of sGC by an NO trap at 22°C in the absence or presenceof nucleotides. sGC was premixed with GMPCPP and�or ATP, or no nucleotide, and excess NO was added via PROLI�NO. Deactivation was initiated bysimultaneous addition of a NO trap [�2(1–217), which removes excess and dissociated NO] and MgGTP. Final concentrations were 48.3 �M �2(1–217), 230 nMsGC-NO, 4.4 mM MgCl2, and 1.9 mM GTP (with or without 625 �M ATP and�or 32 �M GMPCPP). Aliquots were withdrawn at the indicated time points, and thecGMP content of each was measured. Data were fit to an equation describing the accumulation of cGMP (Eq. 1). (C) The decline in enzyme activity as the derivative(Eq. 2) of the accumulation of cGMP shown in B. Data are shown as a percentage of initial activity and are representative of two experiments performed induplicate. (D) Activity of ferrous-nitrosyl sGC formed in the absence or presence of nucleotides. sGC (400 nM) was premixed with 30 �M GMPCPP (G*) and�or1 mM ATP or no nucleotide. Approximately 1 eq of NO was added, and assays were initiated by addition of GTP. Spectra were recorded during each assay.Concentrations of ferrous-nitrosyl sGC were calculated (Fig. 6) for each reaction, and the data show specific activity of the ferrous-nitrosyl species. Basal activityand activity in the presence of excess NO are shown for comparison. (E) Activation of ferrous-nitrosyl sGC by excess NO. ATP (from 375 �M to 3.3 mM) was premixedwith 30 �M GMPCPP (G*) and sGC. The activity of the ferrous-nitrosyl species in the absence (light bars) or presence (dark bars) of excess NO were measured.(F) Activity from E as a percentage of maximum activity.
13066 � www.pnas.org�cgi�doi�10.1073�pnas.0506289102 Cary et al.
GTP to accelerate the dissociation of NO from the sGC heme. Thisfinding suggests that in the presence of ATP and GTP, deactivationof sGC should be extremely slow.
The current binary model states that deactivation of sGC cor-relates with NO dissociation from the heme (Fig. 1, black scheme).Thus, we predicted that, because the NO-heme off-rate is slow inthe presence of ATP and GTP, the deactivation of sGC would becorrespondingly slow. Deactivation studies of sGC under similarconditions as for NO dissociation were carried out. sGC waspremixed with GMPCPP,‡‡ ATP, or both, and excess NO. Deac-tivation assays were initiated by the addition of substrate and a NOtrap, which acts by removing all excess and dissociated NO fromsolution as the assay progresses. In the presence of GMPCPP,deactivation was rapid (t1/2 �7 s), in good agreement withpreviously reported values [t1/2 �4 s (28)]. Surprisingly, thedeactivation of sGC is extremely rapid in every situation, regardlessof nucleotide or NO trap (Fig. 2 B and C; and see Fig. 7, which ispublished as supporting information on the PNAS web site). ATPdoes not slow deactivation of sGC despite the slow NO-hemeoff-rate we observe when ATP is present. The rapid deactivation ofsGC observed in the presence of ATP and GTP, although consis-tent with in vivo observations of sGC deactivation, is inconsistentwith the paradigm that NO binding solely to the heme results in fullactivation. In the presence of ATP and GTP, it is the removal ofnonheme NO that results in the rapid deactivation of sGC.
The �160-fold difference in the rates of NO dissociation andsGC deactivation as described above suggests that NO remainsbound to deactivated sGC. In fact, we were able to isolate deacti-vated sGC and show that the heme remained largely NO-bound(see Supporting Text and Fig. 8, which are published as supportinginformation on the PNAS web site). This finding is consistent witha recent report in which a low-activity species of sGC with NObound to the heme was described. In this report, when excess NOwas removed from sGC, the activity of the resulting ferrous-nitrosylenzyme was 10–20% of full activity (21). Together, these observa-tions indicate that the formation of ferrous-nitrosyl sGC is notalways sufficient for full enzyme activation. Indeed, when wedeactivate sGC in the presence of ATP and GTP, a ferrous-nitrosylspecies with low-activity results. This is contrary to the currentmodel, which maintains that ferrous-nitrosyl sGC is the activatedform of sGC, and that loss of NO from the heme is the mechanismby which deactivation occurs.
Binding of NO to the sGC heme is not sufficient for fullactivation; some other factor must be required for full activation. Inthe report by Russwurm and Koesling (21), the authors show thathigh concentrations of substrate GTP (5 mM) or the cyclasereaction products cGMP (1 mM) or pyrophosphate (PPi) (600 �M),when present with sGC before the addition of NO, result in fullyactive enzyme without excess NO. They concluded that it is theseproducts, not GTP, that are responsible for enabling full activationof sGC by NO. However, the concentration of cGMP used in theirexperiments is orders of magnitude higher than estimated physio-logical concentrations of cGMP [0.1–10 �M (31)]. In light of ourdata above indicating that the effect of GTP on sGC activation byNO is turnover-independent, we confined the experiments reportedhere to more physiological concentrations of GTP when investi-gating the ability of nucleotides to regulate NO activation of sGC.
To determine the activity of the ferrous-nitrosyl species of sGCformed in the absence of excess NO and in the presence of ATP andGTP, a concentration of NO substoichiometric with the sGC hemewas used to form the ferrous-nitrosyl complex. For each experi-ment, the concentration of ferrous-nitrosyl sGC was calculated (Fig.6), and the activity due to that species was determined. When sGC
is premixed with GMPCPP, formation of the ferrous-nitrosyl sGCspecies leads to full activity. If GMPCPP is added after theformation of the ferrous-nitrosyl sGC species, activity is low (datanot shown). When ATP is present with GMPCPP, ATP essentiallyblocks GMPCPP from affecting formation of fully active ferrous-nitrosyl enzyme (Fig. 2D). Thus, in the presence of ATP and GTP,binding of NO to the sGC heme does not fully activate the enzyme,implying that excess NO is required to fully activate the ferrous-nitrosyl sGC.
We next investigated the effect of excess NO on the low-activityferrous-nitrosyl sGC species formed in the presence of ATP andGTP. sGC was premixed with a fixed concentration of GMPCPPand increasing concentrations of ATP. Activities were then deter-mined in the presence of substoichiometric or excess NO. As before,the concentration of ferrous-nitrosyl sGC was calculated, and theactivity due to that species was determined. With substoichiometricNO, when no nucleotide is premixed with sGC, the activity of theferrous-nitrosyl enzyme is low (Fig. 2 E and F). PremixingGMPCPP with sGC leads to a ferrous-nitrosyl species with fullactivity. As the concentration of ATP increases, the ability ofGMPCPP to effect formation of the fully active ferrous-nitrosylspecies is progressively blocked, resulting in an increasing propor-tion of low-activity ferrous-nitrosyl sGC. Full activation of thislow-activity species is only possible by the addition of excess NO.
The data presented thus far suggest a previously uncharacterizedmodel for activation and deactivation of sGC (Fig. 3). Underphysiological ATP and GTP concentrations, low concentrations ofNO (tonic NO) bind to the sGC heme, resulting in a low-activitystable species. This ferrous-nitrosyl species is sensitized to furtheractivation by additional NO. High concentrations of NO (acuteNO) rapidly and fully activate sGC. As soon as NO concentrationsdrop, the enzyme deactivates, returning to its sensitized ferrous-nitrosyl state. When cellular ATP levels are low, any ferrous-nitrosyl sGC formed in the presence of GTP will be fully active. Itis currently unknown where ATP and GTP bind to exert their effect
‡‡For activity studies, we used the GTP analogue GMPCPP, which is not turned over by sGC,for premixing instead of GTP. Premixing with low concentrations of GMPCPP producesthe same effect as GTP in spectral studies and does not interfere with enzyme activity.
Fig. 3. A previously uncharacterized model for sGC activation and deacti-vation. When ATP and GTP are present (the most likely physiological scenario),the ferrous-nitrosyl species is extremely stable at low NO concentrations andhas low activity. Higher NO concentrations fully activate sGC, albeit tran-siently. When ATP levels drop (such as under conditions of cellular stress), GTPand NO at the heme are sufficient for full enzyme activity, which is alsotransient. Although ATP and GTP are depicted binding to the same or over-lapping sites, it remains possible that the nucleotides bind to separate sites onthe enzyme.
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on sGC activity. A recent modeling and mutational study suggeststhat both ATP and GTP bind to a pseudosubstrate site in thecatalytic domains (22); however, it remains a possibility that ATPand�or GTP might bind elsewhere on the enzyme.
While studying the effect of nucleotides on ferrous-nitrosyl sGC,we noticed that ATP inhibits NO binding to the sGC heme, whereasGTP increases binding. We observed that when a fixed substoi-chiometric concentration of NO is added to sGC (�max 431 nm) inthe presence of increasing concentrations of ATP, less formation ofthe ferrous-nitrosyl species (�max at 399 nm) occurs (Fig. 4A). Theseobservations were unaffected by the presence of a fixed concen-tration of GMPCPP (Fig. 4B). Furthermore, GMPCPP aloneincreases ferrous-nitrosyl formation compared with the nucleotide-free sample (Fig. 4C). To observe the effect of nucleotide onheme–NO binding over time, sGC was mixed with a fixed amountof GTP and increasing concentrations of ATP. NO was then added,and binding was monitored at 4°C. In the absence of nucleotide, asdescribed in Fig. 1 (black scheme), binding of NO to the hemeproceeds through a six-coordinate intermediate, with a �max at 420nm (18). In the presence of GTP, the six-coordinate intermediateis barely evident (Fig. 4D). This finding indicates that conversion ofthe six-coordinate intermediate to the five-coordinate complex isaccelerated by GTP. Similar data have been reported (21). How-ever, when ATP is included with GTP, a peak at 420 nm appears.This peak is indicative of the six-coordinate intermediate andbecomes more prominent as the concentration of ATP is increased(Figs. 4 E–G). Thus, acceleration of the formation of five-coordinate ferrous-nitrosyl sGC by GTP is inhibited by ATP.
The ability of ATP to slow formation of the final heme-NOcomplex and the acceleration of this conversion by GTP correlateswith the effect of each nucleotide on NO dissociation rates.Importantly, ATP blocks any acceleration by GTP of the formationor dissociation of the five-coordinate NO-heme complex. These
observations lead us to propose a biochemical scheme relating NObinding dynamics at the sGC heme with enzyme activity (Fig. 1, redscheme). In the presence of GTP, formation and dissociation of thefinal heme-NO complex are rapid, and the activity of the corre-sponding ferrous-nitrosyl species is high. However, in the presenceof ATP and GTP, heme-NO complex formation and dissociationare slow, and the corresponding ferrous-nitrosyl species has a lowactivity. This low-activity ferrous-nitrosyl species requires nonhemeNO to be fully activated. Physiologically, the most likely scenario isone in which both ATP and GTP are present with sGC; in thisscenario, NO will bind to sGC to form stable low-activity ferrous-nitrosyl complexes sensitized to full activation by subsequent acuteNO signals.
To date, physiological and therapeutic NO signaling studies havebeen designed and interpreted based on the binary model of NOactivation of sGC. Clearly, in the presence of ATP and GTP,NO regulates not one, but two activation states of sGC. At low NOconcentrations, such as the tonic NO production in tissues, the sGCheme forms a stable complex with NO. This ferrous-nitrosyl specieshas a persistent, low activity when ATP and GTP levels are normal.Acute concentrations of NO (typically in the low to mid nanomolar)fully activate ferrous-nitrosyl sGC, and deactivation of fully acti-vated sGC is rapid. Although direct observation of the ligation stateof sGC is not possible in vivo, YC-1, an exogenous activator of sGC(32), might act as a reporter for the presence of the low-activityferrous-nitrosyl species. Previous reports have shown that theeffects of YC-1 on basal and NO-stimulated activities of sGC areminimal, but that it synergistically activates low-activity ferrous-COsGC (33, 34). Because ferrous-nitrosyl sGC has low activity in thepresence of ATP and GTP (vide supra), the effect of YC-1 on theactivity of this species was determined. We found that YC-1 fullyactivates the low-activity ferrous-nitrosyl enzyme (Fig. 5). Further-more, when YC-1 and excess NO are mixed with sGC, rapid
Fig. 4. ATP inhibits the formation of the sGC heme-NO complex. sGC (400 nM) was premixed with no nucleotide, 30 �M GMPCPP, or 30 �M GMPCPP and 0 �M,375 �M, 750 �M, 1.5 mM, or 3.3 mM ATP at 22°C. The same amount of PROLI�NO was added to all samples, which were then analyzed by absorption spectroscopy.Final spectra are shown. The spectra of ferrous sGC and sGC with excess NO (xsNO) are shown for comparison. (A) ATP prevents formation of ferrous-nitrosyl sGC,whereas GMPCPP (G*) increases formation. (B) The experiment shown in A was repeated with GMPCPP (G*) and ATP together. ATP prevents GMPCPP fromincreasing ferrous-nitrosyl formation. (C) GMPCPP (G*) accelerates formation of ferrous-nitrosyl sGC. (D) The effect of nucleotides on the binding of NO to thesGC heme at 4°C. sGC (400 nM) was premixed with 200 �M GTP, 200 �M GTP and 500 �M ATP (E), 200 �M GTP and 3 mM ATP (F), or 3 mM ATP (G). Excess PROLI�NOwas added, and spectra were collected every 12 s to monitor NO-heme binding. The t 0 spectrum is blue, followed by pink, green, and orange for t 12, 24,and 36 s, respectively. Spectra acquired in the absence of nucleotide were the same as those acquired in the presence of 3 mM ATP (data not shown). In thepresence of increasing amounts of ATP, the six-coordinate intermediate (�max at 420 nm) is visible. Little intermediate is observed with GTP alone; ATP blocksGTP from accelerating the formation of the final five-coordinate NO-heme complex.
13068 � www.pnas.org�cgi�doi�10.1073�pnas.0506289102 Cary et al.
deactivation with a NO trap does not occur, as reported (28). Giventhat YC-1 does not affect the rate of dissociation of NO from thesGC heme (S.P.L.C. and M.A.M., unpublished work), we concludethat it is a stable synergistic activator of low-activity ferrous-nitrosylsGC.
Many physiological studies with YC-1 describe it as a NO-independent activator of sGC, or point to its effects as proof thatCO is coactivating sGC. For instance, YC-1 dilates blood vesselswithout exogenous NO or acetylcholine; this YC-1 effect in tissuesis reversed extremely slowly (35). Other studies found that, after anacute NO signal in cerebellar cells, sGC is deactivated almostimmediately to a low-activity ‘‘desensitized’’ state (36, 37). Subse-quent addition of YC-1 fully reactivates this sGC species. Our datasuggest that the use of YC-1 in vivo stimulates cGMP production bysynergizing with the low-activity ferrous-nitrosyl sGC that is likelypresent in tissues. This finding may explain why YC-1 can have sucha potent, long-lived effect on resting tissue or deactivated sGC:low-activity sGC with a stable ferrous-nitrosyl complex will bestably and fully activated by YC-1. We proposed that YC-1 syner-gizes with a low-activity NO-bound species of sGC to mimicactivation by high concentrations of nonheme NO.
The data presented in this article suggest that tonic and acute NOsignal transduction in vivo is due to the two NO activation states weobserve in vitro. The stable low-activity state of sGC and thetransient high-activity state, both of which are regulated by NO,appear to be essential properties of NO�cGMP signaling. A low-activity NO-bound species of sGC was recently observed, butbecause it did not form in the presence of substrate, it wasdiscounted as physiologically irrelevant (21). The data we reporthere show that ATP mediates formation of this low-activity NO-bound species; furthermore, we show that NO dissociates from thisspecies �500 times more slowly than the NO-bound species formedin the presence of GTP alone. It has been proposed that the abilityof ATP to directly affect sGC activity provides the cell with amechanism for linking NO�cGMP signaling with changes in cellularmetabolism (23). Here, we have extended the understanding of howATP affects NO�cGMP signaling, providing insight into the mech-anism of direct ATP regulation of sGC activity in the context ofphysiological nucleotide levels. Furthermore, the studies we de-scribe here on NO-heme association and dissociation, and sGCactivation and deactivation, provide a model consistent with in vivoobservations of the amplitude and duration of tonic and acute sGCactivity. Perhaps the most striking example of tonic and acuteNO�cGMP signaling working in concert is in hippocampal long-term potentiation (LTP) (38). Tonic NO is required before andafter the acute NO signal to achieve LTP. As a behavioral correlateto LTP, blocking the hippocampal NO�cGMP pathway in ratsimmediately after an inhibitory avoidance learning task producesamnesia (39, 40). Furthermore, when YC-1 is administered beforeor after a learning task, it enhances learning and memory (41, 42).Because we show here that NO can affect sGC activity in twofundamentally distinct ways, we believe that NO signaling studies intissues, and the development of therapeutics, must consider thispreviously uncharacterized NO duality.
We thank the members of the Marletta laboratory for discussions andcritical reading of the manuscript. This work was supported by theLaboratory Directed Research and Development Fund of LawrenceBerkeley National Laboratory.
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Fig. 5. YC-1 fully activates low-activity ferrous-nitrosyl sGC. Shown are theactivities of basal sGC, ferrous-nitrosyl sGC, and ferrous-nitrosyl sGC withexcess NO (200 nM sGC in each experiment) with or without 100 �M YC-1 at22°C. Also shown is the effect of YC-1 on the deactivation of sGC (67 nM)activated by excess NO. YC-1 was always added to sGC before PROLI�NO.
Cary et al. PNAS � September 13, 2005 � vol. 102 � no. 37 � 13069
